Interactions between Shh pathway regulators and fetal alcohol exposure in mice

描述(申请人提供)：无前脑畸形(HPE)是一种常见的前脑和面中部出生缺陷，既有环境原因，也有遗传原因。环境因素之一是孕妇在怀孕早期饮酒。此外，在人类HPE中还发现了Sonic hedgehog(Shh)信号通路的突变。突变携带者的HPE表型变化很大，影响范围从前脑和面部中部的严重缺陷到没有临床表现。这种差异的原因尚不清楚，但也可能需要的因素包括额外的遗传影响或环境暴露，在胎儿发育期间。基因-环境的相互作用很可能涉及到像HPE这样的复杂疾病；然而，关于这一点的模型很少。CDO和Boc是细胞表面蛋白，结合Shh并调节发育中胚胎特定区域的信号强度。CDO突变小鼠表现出菌株特异性的HPE：129/Sv背景上的突变小鼠表现出非常轻微的低频HPE，而C57BL/6背景上的突变小鼠在高频下表现出更严重的HPE。BOC突变小鼠无明显效应，但129/Sv CDO；Boc双突变小鼠表现出严重的颅面部HPE特征。我们报道129/Sv小鼠对乙醇诱导的HPE具有抵抗力；然而，在宫内暴露于乙醇的129/Sv CDO突变胚胎中，80%的胚胎表现出强烈的颅面部HPE，并与CDO；Boc双重突变株有惊人的相似之处。此外，乙醇处理的CDO突变胚胎的前脑Shh表达显著降低。因此，CDO的丢失和胎儿乙醇暴露可以协同作用产生HPE；这一事实，加上乙醇处理的CDO突变胚胎与CDO如此相似；Boc双突变体，认为CDO的丢失和乙醇暴露对Shh信号具有协同作用。肢体和手指缺陷也与人类胎儿酒精暴露有关，在宫内接触C57BL/6胚胎酒精会导致四肢和手指后部畸形。所有肢体和手指的后方花纹都需要Shh。丢失Boc，但不是CDO，会在遗传敏感的背景下导致严重的手指缺陷。假设与129/Sv CDO突变小鼠和HPE相似，129/Sv Boc突变小鼠将对乙醇引起的肢体/手指畸形致敏。关于乙醇如何扰乱已知的肢体/手指图案调节因子的信息相对较少，而对经乙醇处理的C57BL/6和129/Sv小鼠的发育遗传学分析将有助于揭示这一过程。本研究的目的是：1)分析酒精诱导的129/Sv CDO突变小鼠的HPE；2)确定酒精诱导的129/Sv CDO突变小鼠前脑Shh表达下调的机制；3)分析Shh通路在乙醇诱导的肢体/手指图形缺陷中的作用。这些研究涉及分析一种新开发的基因-环境相互作用模型，该模型在一种常见的出生缺陷中存在明确的发育突变和酒精之间的相互作用。这些信息可能会对公共卫生产生重要影响，并最终可能有助于对突变携带者的咨询。 公共卫生相关性：孕妇在怀孕早期饮酒可能导致出生缺陷，这可能受到人类群体遗传变异的影响。这项应用专注于一种小鼠模型，在该模型中，宫内酒精暴露和一种特定的突变组合会导致前脑和面部中部毁灭性的常见出生缺陷，称为全前脑。建议对这一模型进行研究，并将其扩展到更多与酒精相关的出生缺陷，目的是揭示遗传易感性如何影响胎儿酒精暴露的结果。

DESCRIPTION (provided by applicant): Holoprosencephaly (HPE) is a common birth defect of the forebrain and midface with both environmental and genetic causes. Among the environmental factors is maternal alcohol consumption during early pregnancy. In addition, mutations in the Sonic hedgehog (Shh) signaling pathway have been identified in human HPE. The HPE phenotype of mutation carriers is highly variable, with the spectrum of effects ranging in a continuum from severe defects in the forebrain and midface to no clinical manifestation. The reason for this variability is un- known, but factors that may also be required include additional genetic influences or environmental exposures, during fetal development. Gene-environment interactions are likely to be involved in a complex malady like HPE; however, there are few models for this. Cdo and Boc are cell surface proteins that bind Shh and regulate pathway signaling strength in specific regions of the developing embryo. Cdo mutant mice display HPE with strain-specific severity: mutants on the 129/Sv background show very mild HPE with low frequency, while mutants on the C57BL/6 background show more severe forms at high frequency. Boc mutant mice are without overt effect, but 129/Sv Cdo;Boc double-mutants display severe craniofacial HPE features. We report that 129/Sv mice are resistant to ethanol-induced HPE; however, 80% of 129/Sv Cdo mutant embryos exposed in utero to ethanol display strong craniofacial HPE and bear a striking resemblance to Cdo;Boc double mutants. Furthermore, there is a strong reduction of Shh expression in the forebrain of ethanol-treated Cdo mutant embryos. Loss of Cdo and fetal ethanol exposure therefore synergize to produce HPE; this fact, plus that ethanol-treated Cdo mutant embryos are so similar to Cdo;Boc double-mutants, argues that loss of Cdo and ethanol exposure have a synergistic effect on Shh signaling. Limb and digit defects are also associated with human fetal alcohol exposure, and in utero ethanol exposure of C57BL/6 embryos results in malformations of the posterior aspects of limbs and digits. Shh is required for all posterior patterning of limbs and digits. Loss of Boc, but not Cdo, results in severe digit defects on a genetically-sensitized background. It is hypothesized that, similar to 129/Sv Cdo mutant mice and HPE, 129/Sv Boc mutant mice will be sensitized to ethanol-induced limb/digit anomalies. There is relatively little information available on how ethanol disrupts the known regulators of limb/digit patterning, and developmental genetic analyses of ethanol-treated C57BL/6 and 129/Sv mice will shed light on this process. The aims of this proposal are: 1) to analyze ethanol-induced HPE in 129/Sv Cdo mutant mice; 2) to identify mechanisms of ethanol-induced down-regulation of Shh expression in the forebrains of 129/Sv Cdo mutant mice; and 3) to analyze the role of the Shh pathway in ethanol-induced defects in limb/digit patterning. These studies involve analysis of a newly developed model of gene-environment interaction between a defined developmental mutation and ethanol in a common birth defect. Such information may have important public health impact and could ultimately aid in the counseling of mutation carriers. PUBLIC HEALTH RELEVANCE: Maternal alcohol consumption during early pregnancy can result in birth defects, and this may be influenced by genetic variation in the human population. This application focuses on a mouse model in which a combination of in utero alcohol exposure and a specific mutation leads to a devastating and common birth defect of the forebrain and midface, called holoprosencephaly. Studies on this model, and its extension to additional ethanol-associated birth defects, are proposed, with the goal of shedding light on how genetic susceptibility may influence the outcome of fetal alcohol exposure.